NSABB July 1, 2005 Synthetic Genomics From Reading to Writing the Genetic Code Genes are the design components of the future Ten years ago this month Microbial Abundance . Microbes make up roughly ½ of the Earth’s biomass . 6 x 1030 microbes over entire Earth . Animals make up 1/1000th of Earth’s total biomass . Each ml of sea water has one million bacteria and 10 million viruses www.sorcerer2expedition.org Sorcerer II Global Ocean Survey: Nova Scotia through Galapagos Islands Sargasso Sea Pilot Study 100 Million letters of genetic code every 24 hours 454 Genome Sequencing System . Up to 100x throughput over Fluorescent Sequencing: - 20 megabases/4 hr instrument run (20-32 high Q) Current vs 454 Platform 3730 454 Bases per day 945K 100-200 million Runs per day 12 5 Bases per run 78.7K 20-40 million Average RL 820bp 125bp Cost ~$0.0012/base $0.00025/base ( $1.20/Kb) ($0.25/Kb) Marine Microbe Sequencing Project . $9M Funded by Gordon and Betty Moore Foundation Sequence, Assemble and Auto-Annotate up to 130 Marine Microbes Add to the current (10-20) set of sequenced Marine Microbes - 1000% increase Synergy with Sorcerer Expedition Sorcerer II Expedition and Moore Foundation Isolated Organisms 1 4 11 1 4 1 2 3 Canada 8 1 7 1 1 1 12 4 2 2 1 Bermuda Mexico 3 1 1 1 2 Honduras1 Panama 2 2 1 Cocos Islands 1 1 French 2 Galapagos Tonga Polynesia Islands Vanuatu 1 Fiji Cook New Islands 1Caledonia Australia 1 1 1 1 Sampled stations Planned stations Number of organisms isolated from each region for the Moore Foundation Program Aerosol sample collection . Samples 1360 m3 air in 24 hrs (58 m3/hr) . Average 1 X 109 bacteria/filter/day . Multi-day sample averages known diurnal variation in microbial composition Preliminary Analysis: Nova Scotia through Galapagos Synthetic Genomics Design and construction of genomes from scratch Chemical synthesis of DNA: from genes to genomes 1000000 Small bacteria Large viruses 100000 Size PKS gene of cluster project 10000 phiX (bp) Published Projected poliovirus 1000 gene+plasmid 100 tRNA 10 '75 '80 '85 '90 '95 '00 '05 '10 Year of publication M. genitalium: Living Organism With the Smallest Genome Whole genome phylogeny of Firmicutes M. pnemoniae clade M. hominis clade Spirolasma-M.mycoides clade subgroups Mycoplasma Phytoplasma asteris OY strain Each partition has bootstrap support at least 97% The core mycoplasma genome is the set of genes common to all 13 complete sequences At least 36 genes Expanded core in expanded core of 310 genes (90 are non-essential because of non- based on gene orthologous gene disruption studies displacements 173 genes 220 core genes common to w/o obligate all 13 Intracellular parasite Phytoplasma Construction of an Artificial Chromosome Oligonucleotides Taq Polymerase Assembled Gene PCR Products ~ 10 Kbp Artificial Chromosome Rapid gene synthesis from oligonucleotides Hamilton Smith, Clyde Hutchison, Cindi Pfannkoch, Craig Venter Phi X-174 This Software Builds Its Own Hardware From Reading To Writing: What can be made and when? . Any sequenced viral genome including select agents can be made today . Designer viruses” are over a decade away . Prokaryotes (bacteria): 2 years . Single-cell eukaryotes: Within 10 years Construction of an Artificial Chromosome Oligonucleotides Taq Polymerase Assembled Gene PCR Products ~ 10 Kbp Artificial Chromosome How do we connect many small synthetic genome fragments into a single circular chromosome? HOMOLOGOUS RECOMBINATION IN VITRO In vitro Deinococcus radiodurans Courtesy Michael Daly homologous Hours after recovery recombination 0 2 4 2 O 2 w/o H 10 mM 20 mM 30 mM 40 mM 10 mM 20 mM 30 mM 40 mM 10 mM 20 mM 30 mM 40 mM DNA maker DNA • Develop an in vitro D. radiodurans recombination system • Express and characterize recombination proteins 48.5 kb GroEL DrRecA DrSsb D. radiodurans: The Ultimate DNA Assembly Machine 0.013 DSB/Gy With David Schwarz, University of Wisconsin, Madison Combinatorial Genomics . Genes can be rapidly screened via cassette based construction of thousands to millions of genomes/day . Selection through screening for Chemical production Viability Hydrogen production etc. Genome transplantation Ribosomes + enzymes Synthetic Nuclear + = material Synthetic Ribosomes + genome enzymes Sugars CO2 Synthetic Cell Protein Biopolymers Methane CH4 (CH2) n CO Synthetic Cell Hydrogen Methane CH4 Photosynthetic production of hydrogen in cyanobacteria NADP+ NADPH Calvin cycle FNR hydrogenase fixation + CO2 H2 H + Ferredoxin- ATP H+ ADP PSII 2H+ ATP Synthase Fx Qa Qb cyt b6f A1 Pheo QH2 PSI P690 Q Q A0 FeS Mn4 2H+ PC/cytc H+ + - H2O O2 + 2H + e Some Potential benefits . Basic biology . Human and animal health • Study evolution • Vaccines (research and • Understand requirements applications) for life • Gene therapy • Confirm sequences • Phage-based antibiotics • Drugs . Energy . New materials • e.g., Renewable hydrogen • Bioplastics (a JCVI project funded by DoE Office of Science) Future of Engineered Species . Designed and engineered species could replace petro-chemical industry . Will be a source of future food . A source of energy . The basis of bioremediation Regulation vs Good Scientific Standards . We Postponed Our research for Ethical/Policy Review in 1999 Review indicated acceptable to proceed with minimal genome efforts* . Good Steward Standard Responsible lab precautions Lab Bio Safety Level 3 for early stages No human pathogens/No human genomes No organism survival outside lab Engineer out pathogenesis and self evolution Open communication with non-science communities . Tremendous Opportunity for Good Carbon neutral energy sources Environmental remediation Carbon sequestration Vaccine and other opportunities * “Genetics: Ethical Considerations in Synthesizing a Minimal Genome” Science, 286, p. 2087 Ethics and policy studies . Ethical and religious concerns surrounding work on minimal genomes (1999) • Ethics of Genomics group considered the implications of determining and/or synthesizing a minimal genome • Identified specific areas of concern, but determined that the research overall does not cross any bioethics boundaries – Cho et al., 1999. Science 286: 2087-2090. Societal implications (ongoing) • Synthetic Genomics: Risks and Benefits for Science and Society • 15-month study, funded by the Sloan Foundation • Focus on bioterrorism and environment, health, and safety • Partners – Policy Center, Venter Institute – Homeland Security Program, Center for Strategic & International Studies – Synthetic Biology Group, Massachusetts Institute of Technology.